My Northerns won't work!

[Cathy Sprankle]

After 5+ years of running Northerns pretty much without any trouble, a few
months ago they stopped working for me.  I am getting a hybridization
signal on my blots, just not much of one (maybe 25% of what I should be
seeing).  RNA appears to be intact (the signals I'm getting are bands, not
smears), and hybridization with a control probe confirmed that the problem
is with the blots themselves rather than the hybridization conditions or
procedure or the probes I've been using.  I compared UV crosslinked blots
to baked blots, no difference.  Also compared two different packs of
Hybond N, again no difference.  Where this leaves me is:

--something is chewing up my RNA during the gel washes, during the
transfer, or after the transfer, or
--something is wrong with my transfer (old-fashioned upward capillary blot
procedure).

I would appreciate suggestions about where I should go from here.  Thanks
very much.

Cathy

-- 
Catherine Sprankle
CIIT
e-mail:  sprankle at ciit.org
Opinions expressed (such as they are!) are strictly mine and do
not reflect those of CIIT.